Cell cycle checkpoints react to the presence of damaged DNA, and organize the cellular response to the damage. Without intact checkpoint control systems, genome stability is at risk, and the potential for accumulating mutations in critical growth control genes is significantly enhanced. Although much is known about how checkpoints regulate the cell cycle and organize DNA repair systems, comparatively little is known about how checkpoints are activated by damaged DNA in the first place. An emerging idea in the checkpoint field is that, during S phase, some forms of damage are sensed by replication forks. If so, then a protein that is required for both replication and checkpoint control represents a good candidate to provide the elusive damage sensing activity. The Cut5 protein in fission yeast (Dpb11 in budding yeast) fulfills this requirement, as it is required for both DNA replication and the checkpoint response to stalled replication forks. Additionally, Dpb11 has been shown to play a role in maintenance of genome stability, as hypomorphic alleles of DPB11 cause a dramatic increase in chromosomal rearrangements even though the cells are viable. The closest match to Cut5/Dpb11 amongst vertebrates is the Mus101 protein family, of which the human TopBP1 is a member. Despite its similarity to a yeast protein with a proven role in checkpoint control, Mus101/TopBP1 remains poorly characterized. In order to understand more about the role of Mus101 in replication and checkpoint control, my laboratory has characterized Mus101 activity in both the biochemically tractable Xenopus egg extract system and the genetically tractable nematode C. elegans. We have found that depletion of Mus101 from Xenopus egg extracts blocks both DNA replication, and activation of the DNA damage checkpoint. Furthermore, depletion of the C. elegans mus-101 ortholog results in embryonic lethality and, under hypomorphic conditions, sensitivity to DNA damage. These findings establish that Mus101 is the vertebrate counterpart to Cut5/Dpb11. The goal of this proposal is to combine biochemical analysis of Mus101 in Xenopus egg extracts with genetic analysis in C. elegans to fully describe the Mus101 function in DNA replication, in activation of the DNA damage checkpoint, and in maintenance of genome stability.